Abstract 13742: IQGAP1, a VEGFR2 Scaffold Protein, Sequesters c-Cbl and Regulates VEGF Receptor 2 Stability: Role In VEGF-Induced Angiogenesis
We previously identified IQGAP1 as a novel VEGF receptor 2 (VEGFR2) binding scaffold protein, which plays a critical role in reactive oxygen species (ROS)-dependent VEGF signaling involved in endothelial proliferation and migration. However, a role of IQGAP1 in regulating VEGFR2 function remains unknown. Here we show that IQGAP1 protects the VEGFR2 from the negative regulatory activity of the c-Cbl E3 ubiquitin ligase in human endothelial cells. Western analysis shows that depletion of IQGAP1 with siRNA enhances VEGFR2 protein degradation after 15 min of VEGF stimulation (55%), while overexpression of IQGAP1 prevents VEGF-induced downregulation of receptor. Cell surface biotinylation and immunofluorescence experiments show that knockdown of IQGAP1 reduces cell surface expression of VEGFR2 (70%). VEGF-induced downregulation of VEGFR2 is prevented by treatment with proteasomal inhibitors (MG132), but not lysosomal protease inhibitor (chloroquine). Mechanistically, IQGAP1 depletion enhances VEGFR2 ubiquitination (75%). VEGF-induced VEGFR2 downregulation is prevented by overexpression of dominant negative c-Cbl which inhibits ubiquitination. Co-transfection experiments show that IQGAP1 directly binds to both VEGFR2 and c-Cbl. IQGAP1 knockdown promotes VEGFR2-cCbl association, suggesting that IQGAP1-cCbl binding inhibits the binding of Cbl by the VEGFR2 and thus prevents Cbl from catalyzing receptor ubiquitination. As its consequence, IQGAP1 depletion inhibits VEGF-induced phosphorylation of VEGFR-2 (78%) and AKT (82%) as well as endothelial proliferation. In vivo, IQGAP1 deficient mice show reduced expression of VEGFR2 protein (53%) and impaired angiogenesis in hindlimb ischemia model as compared with wild type mice. In summary, IQGAP1 functions as a scaffold to sequester c-Cbl and thus prevents VEGFR2 degradation, thereby promoting VEGFR2 stabilization and signaling linked to angiogenesis. Thus, targeting IQGAP1-c-Cbl interaction is a novel therapeutic strategy for angiogenesis-dependent cardiovascular disease.
- © 2011 by American Heart Association, Inc.